Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

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Disease processes that are currently among the leading causes of death now require much more than just a stethoscope for diagnosis and a pill for treatment. The next generation of therapeutics needs to possess a degree of intelligence; the ability to sense and respond to their environment. Biomedical hydrogels have the ability to sense and respond to external stimulus and with the advent of nanotechnology; these polymers can be fabricated on the same size scale as cellular and sub-cellular processes. Throughout the body gradients in pH are used at the cellular level to regulate processes such nutrient transport and to fight infection. Sites of damage or disease within the body are associated with both a change in pH and abnormal nanoporous vasculature. pH-Responsive microgels and nanogels are small enough to access these locations within the body, sense the change in environment, and locally release a therapeutic agent In this work heterogeneous, photoinitiated free radical polymerizations were developed to synthesize novel pH-responsive microgels and nanogels that could be loaded with macromolecular therapeutics and could respond to either a basic or acidic change in pH. A novel photo-dispersion polymerization scheme was developed to synthesize poly(ethylene glycol) grafted poly(methacrylic acid) (P(MAA-g-PEG)) polycomplexation gels for oral protein delivery. These ranged in size from 100- 300 nm in diameter and could swell up to a 17-fold increase in volume, in response to a rise in pH. This property allowed them to protect insulin at low pH and release the protein at neutral pH. In this way the carriers could be used to transport proteins through the stomach to the small intestine for absorption. A novel photo-emulsion polymerization scheme was developed to synthesize poly(ethylene glycol) grafted poly[2-(diethylamino)ethyl methacrylate] nanogels, between 70-150 nm in diameter. These could swell up to a 22-fold increase in volume, in response to a drop in pH. These nanostructures were able to successfully target clathrin-dependent endocytosis and deliver macromolecules to the cytosol. / text

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Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009. / Committee Chair: Garcia, Andres; Committee Member: Babensee, Julia; Committee Member: Bellamkonda, Ravi; Committee Member: Lyon, Andrew; Committee Member: Temenoff, Johnna. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Oligonucleotides are popular recognition-based biomaterials assembly and disassembly tools due to their specificity and ease of control. Their susceptibility to degradation by nucleases and false positive signals under certain conditions, however, has led to great interest in chemically modified oligonucleotides such as locked nucleic acids (LNA) that enhance both nuclease resistance and target specificity. This dissertation extends prior work with DNA sequences to investigate incorporating locked nucleic acid (LNA), a synthetic oligonucleotide, in isothermal colloidal assembly and disassembly schemes as well as on hybridization kinetics between single-stranded and double-stranded probes immobilized on microspheres. Incorporation of LNA nucleotides into DNA sequences is of particular interest as a means of enhancing the performance of DNA in a biomaterials context due to the increased resistance of LNA to nuclease degradation, its greater intrinsic affinity for oligonucleotide targets, and low cytotoxicity effects. The effects of LNA modification, target sequence length, sequence fidelity, and salt concentration are key variables explored. After providing an overview of DNA and its properties, synthetic oligonucleotides, colloidal particles, and previous applications of DNA and LNA in colloidal assembly schemes, this work then discusses the selection and characteristics of appropriate pairs of hybridization partners for reversible colloidal assembly scenarios. A comparative investigation of the in situ primary hybridization kinetics between select LNA or DNA targets and single-stranded probes immobilized on colloidal surfaces is performed. To support the disassembly studies, the in situ competitive displacement kinetics of hybridized LNA primary targets by either LNA or DNA secondary targets is discussed. For these in situ studies, flow cytometry was used to quantify the hybridization reactions as they occur on microsphere surfaces. While comparable rate constants were typically observed between target and single-stranded probes, LNA typically exhibited more extensive primary and secondary hybridization activity. Optimizing hybridization parameters, such as duplex concentration, sequence fidelity, and LNA content in the probe and target strands, allows for the extent of colloidal disassembly to be tuned, an important step in developing a multifunctional colloid-based biomaterial system.

Over 15 million root canal treatments (RCT) are performed yearly in the United States to treat deep caries and dental pulp infection. This procedure however, removes both the diseased and healthy pulp, leading to tooth devitalization. Furthermore, RCTs are associated with a high incidence of re-infection and dentin fracture, reduced sensitivity and eventual tooth loss. Thus there is an unmet clinical need for alternative endodontic therapies that can preserve tooth vitality and ensure long term dental health. The strategy of vital endodontic therapy explored in this thesis centers on the design of a bioactive scaffold that guides host cell homing while providing antibiotic release, in effect harnessing the intrinsic repair potential of the native pulp while simultaneously eliminating residual bacteria that can cause recurrent infection. Specifically, a bioactive polyethylene glycol fibrinogen (PEG-fibrinogen) hydrogel is optimized to support host cell infiltration, maintain dental pulp cell phenotype, and enable pulp regeneration. Ciprofloxacin, a clinically relevant antibiotic for RCT, is incorporated into PEG-fibrinogen to prevent infection. The scaffold and culturing parameters optimized in vitro using either explant or a tooth slice model includes fibrinogen, poly(ethylene glycol) diacrylate (PEGDA) and photoinitiator concentration, as well as cell source and density. In addition, dose-dependent antibiotic effects on both anaerobic bacteria isolated from deep caries and healthy pulp cells are evaluated. The collective findings of this thesis demonstrate that a cell-instructive hydrogel comprised of a fibrinogen backbone and cross-linked with difunctional poly(ethylene glycol) side chains supports pulp cell viability, phenotypic morphology, and host cell migration. Furthermore, increasing pulp cell density promotes cell biosynthesis and a higher fibrinogen concentration is found to enhance collagen deposition. Photoinitiator and PEGDA concentrations have been optimized to enhance hydrogel mechanical properties and gel degradation, while supporting pulp cell phenotype. An optimal antibiotic dosage in the hydrogel has been identified that significantly reduces bacteria count from infected dental pulp without harmful side effects on dental pulp cell phenotype and host cell migration. In summary, this thesis focuses on the design of a bioactive hydrogel-based scaffold with antibiotic release that can induce dental pulp regeneration without the addition of cells and stimuli such as growth factors and minimize post-therapy infection. The innovative scaffold design strategy presented here lays the foundation for the development of vital endodontic therapy that harnesses pulp self-repair and sustains long-term tooth function.

The use of hydrogel nanoparticles, or nanogels, as targeted delivery vehicles to cancer cells was described. The nanogels were synthesized by free radical precipitation polymerization, with poly(N-isopropylmethacrylamide) as the main monomer, and have a core/shell architecture. The nanogels were near 50 nm in radius, contained fluorescein for visualization, and had an amine-containing shell for bioconjugation, making these particles ideal for delivery studies. The nanogels were conjugated with the YSA (YSAYPDSVPMMSC) peptide, which is an ephrin mimic, allowing for uptake by the EphA2 (erythropoietin-producing hepatocellular) receptor. We have delivered YSA-conjugated nanogels to Hey cells and BG-1 cells, as evidenced by fluorescence microscopy. We have shown that the nanogels can encapsulate siGLO Red Transfection Indicator (siGLO) and deliver the siGLO to Hey cells in vitro. After successful delivery of the non-targeting siGLO, we delivered siRNA for knockdown of epidermal growth factor receptor (EGFR). We have shown protein knockdown from 24-120 h after nanogel delivery, as well as knockdown with different siRNA concentrations delivered to the cells. Furthermore, addition of taxol following EGFR knockdown suggests that the chemosensitivity of the Hey cells is increased. Successful in vitro delivery of the nanogels prompted in vivo studies with the nanogels. The nanogels were used to encapsulate silver nanoclusters for potential bioimaging applications. Targeting of the nanogels to MatrigelTM plugs in mice suggest that the particles hold promise as in vivo delivery agents.

Hydrogels, which are polymeric cross-linked networks that swell in aqueous environments, are versatile materials that can contain a variety of chemical functionalities, mechanical properties, and topographical features. Microgels are the stable colloidal form of hydrogel materials that range in size from approximately 100 nm to a few microns in diameter. While they also can exhibit similar properties to those of macrogels, microgels can be used as building blocks in a bottom-up approach to assemble films of higher complexity. In this dissertation, work is focused on understanding the assembly and behavior of microgel thin films as non-fouling surfaces, centrifugally deposited materials, self-healing coatings, and degradable constructs. Non-fouling films were assembled using PEG cross-linked microgels to reduce non-specific protein adsorption and mitigate cellular adhesion. These constructs were assembled in a polyelectrolyte multi-layered fashion, of alternating anionic microgels and cationic linear polymer, to effectively block the substrate from the biological environment and consequently exhibited control over cellular adhesion with the surface. The utility and application of these non-fouling microgel coatings on functional implants was also explored. Centrifugal deposition was used to rapidly generate non-fouling microgel multi-layered interfaces on planar surfaces, and upon closer inspection of the microgel monolayers, it was found that the centrifugally deposited films contained closer-packed microgel assemblies with microgels of smaller footprint size, compared to microgels that are passively adsorbed to the surface. Microgels that are centrifugally deposited may adopt a higher energy chain conformation than passively adsorbed microgels, and this higher energy chain conformation may translate into the multi-layered materials. Nonetheless, the centrifugally deposited non-fouling microgel multi-layered films were found to effectively block macrophage adhesion. Films were also assembled in a polyelectrolyte fashion on soft substrates, and were observed to become significantly damaged under mechanical manipulation (poking, bending, or stretching), but then self-heal upon addition of water. By altering the building blocks of the polyelectrolyte multi-layered films, such as the molecular weight of the polycation between microgel layers or by using anionic rigid spheres as the particle in the assembly, changes in the observed film damage suggest that particle-linear polymer interpenetration and polyvalency likely play an important role in the strength and integrity of the microgel thin films. Fluorescently-labeled microgels were also used to interrogate how the films reorganize in the lateral direction, and these early studies suggest that the microgel multi-layered films reorganize when damaged and also possibly when they are undamaged and simply incubated in an aqueous environment. Additional studies were also conducted on microgels synthesized with a hydrolyzable cross-linker, and by supporting these degradable constructs on substrates, detailed single-particle morphological changes during erosion could be interrogated in complex media such as serum. This work, as a collection, demonstrates the ability to obtain information about microgel thin film assemblies and their behavior using microscopy techniques such as ambient and in liquid atomic force microscopy, brightfield optical microscopy, and fluorescence microscopy. The observations made here illustrate how microgels can be used to fabrication thin films that can be utilized in biological applications (non-fouling, self-healing, and erodable constructs), and how different deposition methods (centrifugal deposition and polyelectrolyte multi-layers) can dictate their behavior.

[Truncated abstract] The synthetic hydrogel poly(2-hydroxyethyl methacrylate) (PHEMA) has been used as a biocompatible biomaterial in ocular devices, such as soft contact lenses, intraocular lenses and an artificial cornea. Due to its favourable properties as an already established (but non-biodegradable) biomaterial, PHEMA is an interesting candidate for use as a material for scaffolds in tissue engineering. A tenant of tissue engineering scaffolds is obtaining the appropriate porous morphology to allow for successful cellular attachment and support. PHEMA hydrogels exhibit varied morphological features, which range from non-porous (homogeneous) to macroporous (heterogeneous) and can be readily obtained by fine-tuning the polymerisation conditions. A desirable feature for matrices that are to be used as tissue supports is the ability to biodegrade in a biological environment. This thesis describes the preparation and enzymatic biodegradation behaviour of novel porous PHEMA hydrogels that have been crosslinked with biodegradable peptide-based crosslinking agents. Peptide-based crosslinking agents were designed to contain two terminal polymerisable groups flanking an internal biodegradable backbone. This backbone was specifically designed to be targeted by the proteolytic enzyme papain. The general design template allowed for the development of a synthetic methodology that was readily implemented for the production of a range of olefin-peptide conjugates. A suite of olefin-peptide conjugates of general structure I were synthesised, characterised and further tested with papain to determine their biodegradation properties. ... The second strategy for producing bioresorbable degradation fragments involved the incorporation of the highly hydrophilic comonomer, poly(ethylene glycol) PEG into the PHEMA backbone. The addition of PEG to PHEMA resulted in the formation of homogeneous hydrogels that had an improved hydrophilicity compared to their heterogeneous PHEMA counterparts. The synthetic conditions for the preparation of PHEMA and PHEMA-co-PEG hydrogels by photoinitiated polymerisation were thoroughly investigated. It was found that the pore morphology and general properties (non-porous to macroporous) of these hydrogels could be controlled by the appropriate choice of polymerisation conditions. The hydrogels were characterised by scanning electron microscopy, thermal gravimetric analysis and differential scanning calorimetry. The peptide-based crosslinking agents were successfully co-polymerised with the HEMA and PEGMA via photoinitiated polymerisation to provide a range of PHEMA and PHEMA-co-PEG hydrogels that displayed both homogeneous and heterogeneous hydrogel properties. The final crosslinked hydrogels were characterised by scanning electron microscopy and were subjected to enzymatic hydrolysis. The PHEMA-peptide conjugate hydrogels proved to be biodegradable, with degradation behaviour dependent on the hydrogel formulation and the length of the peptide-based crosslinking agent.